DNA interstrand cross-links (ICLs) covalently link the Watson and Crick strands of DNA and are extremely cytotoxic. Widely used chemotherapeutics (e.g. nitrogen mustards, cisplatin compounds, mitomycin C) are thought to act through the generation of ICLs. However, tumors almost invariably become resistant to these agents, in part due to upregulation of DNA repair. Importantly, ICLs are also generated by endogenous metabolites (e.g. reactive aldehydes, abasic sites), and failure to repair endogenous ICLs appears to cause human disease. For example, mutation in FANC genes renders cells sensitive to ICLs and causes Fanconi anemia, which is characterized by bone marrow failure and dramatically elevated predisposition to leukemia and other cancers. In addition, mutations in the FAN1 nuclease cause kidney disease. Why two classes of mutations, both of which disrupt ICL repair, cause such different diseases is unknown. To answer this question, it will be critical to understand the molecular functions of the FANC and FAN1 proteins in ICL repair. We have discovered that Xenopus frog egg extracts recapitulate three distinct forms of ICL repair. We previously showed that egg extracts recapitulate replication-coupled ICL repair that depends on the FANC proteins, and we used this approach to show that the FANC proteins lead to DNA incisions that cut-out or unhook the ICL from DNA. In unpublished results, we discovered a second, replication-dependent ICL repair reaction that is however independent of the FANC proteins. In other unpublished results, we recapitulated a third, replication-independent ICL repair reaction that requires the nucleases FAN1 and SNM1A. We hypothesize that the two replication-dependent pathways represent alternative means of repairing ICLs in proliferating tissues, whereas the third pathway is critical in non-proliferating cells. In this proposal, we wil use the power of Xenopus egg extracts to elucidate how ICLs are resolved in the FANC-dependent ICL repair pathway (Aim 1). We will also investigate the mechanism of the FANC-independent ICL repair pathway and determine which of the two replication-dependent pathways are utilized to repair a variety of exogenous and endogenous ICLs (Aim 2). Finally, we will elucidate the roles of FAN1 and SNM1A in replication-independent ICL repair, and we will address whether they perform similar functions in mammalian cells (Aim 3). In summary, this proposal will elucidate the molecular mechanisms of three distinct ICL repair pathways and explore when they are utilized. The potential impact of the work for human health and cancer biology is significant. Small molecule inhibition of factors that promote the novel, FANC-independent ICL repair pathway might re-sensitize FANC-deficient tumors that became resistant to ICLs. Conversely, stimulation of this pathway may have therapeutic benefits for Fanconi anemia patients who are deficient in FANC-dependent ICL repair.